More than 500 million people per year are adversely affected by mosquito-borne diseases such as malaria, lymphatic filariasis and dengue fever. Understanding the physiology of insect vectors, and the molecular interactions between those insects and the pathogens they transmit is critical for controlling these diseases. Viruses, such as Sindbis virus, developed for transient gene expression and gene silencing in insects have proven useful in this type of research. However, the few virus vectors available for this purpose are constrained by narrow host ranges, limited tissue dissemination, inability to express larger genes and human pathogenicity. Our goal is to overcome these problems and develop a highly efficient viral vector system based on a simple RNA virus called Flock House virus (FHV). The virus is a member of the Nodaviridae family of insect viruses. The adaptability of this virus to different hosts, its simple genome organization and non-pathogenecity to humans make FHV an attractive virus for vector development. The 4.5 kb genome of FHV consists of two positive sense single stranded RNAs, which are packaged into a coat protein with no envelope. Although of insect origin, FHV can overcome the kingdom barrier and replicate in plants and yeast. FHV has not previously been shown to multiply in mosquitoes and other insect disease vectors. We have demonstrated that FHV replicates in vitro in cultured mosquito cells as well as in vivo in four mosquito genera, Aedes, Cu/ex, Anopheles and Armigeres. We have expressed the reporter gene GFP in Drosophila ceils and in mosquitoes using FHV vectors generated in this laboratory. We have also shown that FHV replicates vigorously in tsetse flies. We will optimize existing, and develop new FHV vectors to extend its ability to infect medically important insects and express genes of interest. Specifically, we will (1) determine optimum conditions for the growth (both in vitro and in vivo) of FHV in mosquitoes and other insects that transmit diseases including tsetse flies, sand flies and kissing bugs (2) improve the existing FHV-based vectors so that they are capable of expressing or silencing one or multiples gene(s) in insects, and (3) silence two genes that belong to the family of serine proteases from the human malaria vector Anopheles gambiae, and assess their role in melanization. We expect that FHV vectors will be valuable tools for introducing or silencing genes in vivo in medically important insects in order to elucidate the function of genes with various physiological roles. These molecular tools will become increasingly more important as whole organism genome sequencing efforts rapidly progress.